The intracellular synthesis and maturation of the hemagglutinin-neuraminidase (HN) protein of human parainfluenza virus type 3 (PIV3) was investigated with regard to the kinetics of folding and the formation of homooligomers that constitute the HN virion spike. Folding of the HN protein, as monitored by the acquisition of immunoreactivity with conformation-dependent antibodies and by electrophoretic mobility during nonreducing SDS-PAGE, was found to have a half-time of approximately 15 min. Post-infection cotton rat sera and HN-specific murine monoclonal antibodies (mAbs) from a previously-described panel showed little or no reactivity with newly-synthesized, unfolded material, illustrating the conformational nature of the major HN epitopes. The HN protein was shown to form homotetramers by chemical cross-linking and sucrose sedimentation analysis. In all previously studied viral systems, glycoprotein analyzed following a short labeling pulse was present primarily as unfolded monomers which oligomerized only after the monomers had folded into essentially mature conformations. In contrast, for the PIV3 HN protein, newly-synthesized intracellular protein was found exclusively in oligomers which were indistinguishable with regard to sedimentation profile and stability from those of the mature protein. We speculate that oligomerization might be cotranslational and might be related to the type II membrane topography of HN. These studies provide an overview of the time-course of folding and oligomerization of the PIV3 HN protein, demonstrate the conformation-dependence of the major HN epitopes, and illustrate an exception to the previous rule that folding of glycoprotein monomers is a prerequisite to their association into oligomers.